Method for controlling cancer metastasis or cancer cell migration by modulating the cellular level of lysyl trna synthetase

ABSTRACT

The present invention relates to a novel function of lysyl tRNA synthetase, that is, lysyl tRNA synthetase interacts with 67LR through translocation of KRS into plasma membrane, and so enhances tumor (or cancer) cell migration, thereby having an effect on cancer metastasis. More specifically, it relates to method for controlling cancer metastasis or cancer cell migration by modulating an cellular level of lysyl tRNA synthetase, an use of an expression vector comprising a construct inhibiting KRS expression for preventing or treating cancer, an use of an agent inhibiting KRS activity for preventing or treating cancer, a method for screening an agent which modulates cancer metastasis or cancer cell migration, a method for screening an agent inhibiting an interaction between KRS and 67LR. Accordingly, cancer metastasis and cancer cell migration may be controlled using the inventive KRS, further the cellular metabolism related to laminin receptor (67LR) of plasma membrane may be controlled. The relationship between KRS and laminin receptor disclosed in the present invention may be very useful for treatment, prevention and/or diagnosis of various disease related to thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.13/059,006 filed on Feb. 14, 2011, which is a national phase ofInternational Application No. PCT/KR2008/004785 filed on Aug. 18, 2008.The applications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a novel function of lysyl tRNAsynthease. More specifically, it relates to a method for controllingcancer metastasis or cancer cell migration by modulating an cellularlevel of lysyl tRNA synthetase, an use of an expression vectorcomprising a construct inhibiting KRS expression for preventing ortreating cancer, an use of an agent inhibiting KRS activity forpreventing or treating cancer, a method for screening an agent whichmodulates cancer metastasis or cancer cell migration, a method forscreening an agent inhibiting an interaction between KRS and 67LR.

BACKGROUND ART

A tumor is developed by uncontrollable disordered abnormal cellproliferation. Especially, if this tumor shows a destructive growth,invasiveness and metastasis, it is regarded as a malignant cancer.Invasiveness is a character to infiltrate or destroy surroundingtissues, and in particular, a basal layer forming a boundary of tissuesis destroyed by the character, resulting in the local spread andsometimes inflow of a tumor through circulatory system. Metastasis meansthe spread of tumor cells from the original birthplace to other areasthrough lymphatic or blood vessels. In a broad sense, metastasis alsomeans the direct extension of tumor cells through serous body cavity orother space.

These days, surgical operation, radiotherapy and chemotherapy are widelyused for the treatment of cancer singly or jointly. The surgicaloperation is a way to remove diseased tissues. Thus, tumors in specificregions such as breast, colon and skin can be effectively removed by thesurgical operation. However, a tumor in vertebra or dispersive tumorlike leukemia cannot be properly treated by the surgical operation.

Chemotherapy blocks cell replication or metabolism, and has been usedfor the treatment of breast cancer, lung cancer and testicular cancer.Though, patients with cancers who have been treated by chemotherapy haveseriously suffered from the side effects of systemic chemotherapy.Motion sickness and vomiting are common but serious examples of all. Theside effects of chemotherapy can even affect the life of a patient sincethey might drop the adaptability of a patient rapidly. Besides, DLT(Dose Limiting Toxicity) is also one of major side effects ofchemotherapy, which draws a careful attention in the administration of amedicine. Mucositis is an example of DLT against anticancer agents suchas 5-fluorouracil which is an antimetabolic cytotoxic agent, andmethotrexate, and anticancer antibiotics like doxorubicin. If a patientsuffers seriously from such side effects of chemotherapy, he or sheshould be hospitalized and given an anodyne for reducing pain. So, sideeffects of chemotherapy and radiotherapy are the biggest problem for thetreatment of cancer patients.

Gene therapy is a method to treat or prevent diseases caused by thegenetic variation in human cells, for example various genetic disorders,cancers, cardiovascular diseases, infective diseases, and auto-immunediseases, by taking advantage of DNA recombination technique, that is, atherapeutic gene is inserted into a patient to correct genetic defect orto promote or add functions of cells. More precisely, gene therapy is totreat a disease by sending a therapeutic gene to a target organ in orderto induce the expression of therapeutic or normal protein in damagedcells. Gene therapy has advantages such as excellent specificity andimprovement of recovery rate and speed, which are difficult to beregulated by other medicine, which enables long-term administration.Gene therapy is not for treating symptoms of a disease but for curing oreliminating the cause of the disease. For the success of the therapy, itis important to deliver a therapeutic gene to a target cell to improveits expression rate, which is essential technique in gene therapy.

A gene carrier is a necessary mediator for the insertion of atherapeutic gene to a target cell. An ideal gene carrier has to be noharmful for human, mass-produced, has to carry a gene to a target cellefficiently and has to express the gene continuously. Preparation of agene carrier is a core technique in gene therapy. Most representativegene carriers widely used for gene therapy today are viral carriers suchas adenovirus, adeno-associated virus, retrovirus and non-viral carrierssuch as liposome, polyethyleneamine, etc.

As gene therapy strategies for controlling tumor cells, methods of usinga tumor suppressor gene, using a replication-competent oncolytic virus,using a suicide gene and using an immunoregulatory gene, etc, have beenused. The method of using a tumor suppressor gene is to treat cancer bydelivering a tumor suppressor gene such as p53 into a patientspecifically, where the gene is defected or deformed. In addition, themethod of using a replication-competent oncolytic virus is to treatcancer by exploiting the damaged activity of tumor suppressor gene intumor tissues by transferring a viral gene carrier that is able to begrowing specifically in tumor cells to a human body. These two methodsare the strategies to kill tumor cells directly. Alternately, the methodof using a suicide gene is included in this category. A representativeexample of a suicide gene therapy is to treat disease by delivering aHSV-TK gene and chemical anticancer agents such as ganciclovir, whichcan induce death of tumor cells. On the contrary, the method tointroduce an immunoregulatory gene is a kind of indirect treatmentstrategies, which carries one or more of the genes such as interleukin12, interleukin 4, interleukin 7, gamma-interferon and tumor necrosisfactor, etc, into a living body in order to provoke T cells to recognizetumor cells or induce apoptosis by blocking a tumor developing protein.On the other hand, the method to kill tumor cells by blocking nutrientsupply by expressing angiogenesis inhibiting factors such as angiostatinor endostatin, etc, is also included in the category of indirecttreatment strategies.

Metastatic spread is a critical determinant for the lethality of cancer.67 kDa laminin receptor (67LR) is non-integrin type receptor embedded inplasma membrane and associated with cancer invasion and metastasis(Nelson, J. et al. The 67 kDa laminin receptor: structure, function androle in disease. Biosci. Rep. 28, 33-48 (2008)). 67LR is generated fromdimerization of its 37 kDa precursor (37LRP) although molecular detailof this conversion process is not understood. 37LRP is identical toribosomal subunit p40 that is involved in the formation of polysome(Auth, D. & Brawerman, G. A 33-kDa polypeptide with homology to thelaminin receptor: component of translation machinery. Proc. Natl. Acad.Sci. USA 89, 4368-4372 (1992)). 67LR is often observed at high level ina variety of cancers (Nelson, J. et al. The 67 kDa laminin receptor:structure, function and role in disease. Biosci. Rep. 28, 33-48 (2008);Menard, S., Castronovo, V., Tagliabue, E. & Sobel, M. E. New insightsinto the metastasis-associated 67 kD laminin receptor. J. Cell. Biochem.67, 155-165 (1997)). However, the regulator and molecular mechanism forthe membrane residency of 67LR have not been determined yet. In thiswork, the present inventors found that lysyl-tRNA synthetase (KRS)enhances cell migration and cancer metastasis by stabilizing 67LR atplasma membrane.

KRS belongs to aminoacyl-tRNA synthetases (ARSs) that ligate theircognate amino acids and tRNAs for protein synthesis. These ancientenzymes show pleiotropic functions in addition to their catalyticactivities (Park, S. G., Ewalt, K. L. & Kim, S. Functional expansion ofaminoacyl-tRNA synthetases and their interacting factors: newperspectives on housekeepers. Trends Biochem. Sci. 30, 569-574 (2005)).Besides, several mammalian ARSs including KRS form a macromolecularcomplex (Lee, S. W., Cho, B. H., Park, S. G. & Kim, S Aminoacyl-tRNAsynthetase complexes: beyond translation. J. Cell. Sci. 117, 3725-3734(2004); Han, J. M, Kim, J. Y. & Kim, S. Molecular network and functionalimplications of macromolecular tRNA synthetase complex. Biochem.Biophys. Res. Commun. 303, 985-993 (2003)), which serve as molecularreservoir (Ray, P. S., Arif, A. & Fox, P. Macromolecular complexes asdepots for releasable regulatory proteins. Trends Biochem. Sci. 32,158-164 (2007)), to control multiple functions of the componentproteins. Human KRS contains unique N-terminal extension involved in theinteractions with RNA and other proteins (Rho, S. B. et al. Geneticdissection of protein-protein interactions in multi-tRNA synthetasecomplex. Proc. Natl. Sci. Acad. USA 96, 4488-4493 (1999); Francin, M.,Kaminska, M., Kerj an, P. & Mirande. M. The N-terminal domain ofmammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain. J.Biol. Chem. 277, 1762-1769 (2002)).

SUMMARY

To determine the significance of this peptide in relation to thefunctional versatility of human KRS, the present inventors isolated theN-terminal 116 aa peptide of human KRS and used it as the bait for thescreening of its binding proteins from HeLa cell cDNA library usingyeast two-hybrid system. From the screening, the present inventorsidentified 37LRP/p40 as one of the potential binding proteins andinvestigated the functional implication for the interaction between KRSand laminin receptor in this work. To determine the significance of thispeptide in relation to the functional versatility of human KRS, thepresent inventors isolated the N-terminal 116 amino acids peptide ofhuman KRS and used it as the bait for the screening of its bindingproteins from HeLa cell cDNA library using yeast two-hybrid system. Fromthe screening, the present inventors identified 37LRP/p40 as one of thepotential binding proteins and investigated the functional implicationfor the interaction between KRS and laminin receptor in this work.

As a result, the present inventors disclosed that lysyl-t-RNA-synthetase(KRS) enhances cell migration and tumor metastasis by stabilizing 67LRin a plasma membrane to have an effect on cancer metastasis or cancercell migration through a laminin receptor in the plasma membrane,thereby completing the present invention.

An object of the present invention is to provide a novel use oflysyl-t-RNA-synthetase regarding cancer metastasis or cancer cellmigration.

To achieve the above object, the present invention provides a method forcontrolling cancer metastasis by modulating a cellular level of lysyltRNA synthetase.

To achieve another object, the present invention provides a method forcontrolling cancer cell migration by modulating a cellular level oflysyl tRNA synthetase.

To achieve still another object, the present invention provides acomposition for preventing and treating cancer comprising an expressionvector comprising a promoter and a polynucleotide operably linkedthereto, or an antibody against KRS as an effective ingredient, whereinthe polynucleotide is encoding antisense RNA or siRNA against the KRSpolynucleotide.

To achieve still another object, the present invention provides a methodfor preventing and treating cancer comprising administering to a subjectin need thereof an effective amount of an expression vector comprising apromoter and a polynucleotide operably linked thereto, or an antibodyagainst KRS, wherein the polynucleotide is encoding antisense RNA orsiRNA against the KRS polynucleotide.

To achieve still another object, the present invention provides a use ofan expression vector comprising a promoter and a polynucleotide operablylinked thereto, or an antibody against KRS for preparation ananti-cancer agent, wherein the polynucleotide is encoding antisense RNAor siRNA against the KRS polynucleotide.

To achieve still another object, the present invention provides acomposition for preventing and treating cancer comprising an agentinhibiting KRS activity as an active ingredient.

To achieve still another object, the present invention provides a methodfor preventing and treating cancer comprising administering to a subjectin need thereof an effective amount of an agent inhibiting KRS activity.

To achieve still another object, the present invention provides an useof an agent inhibiting KRS activity for preparation a cancer therapeuticagent.

In another aspect, the present invention provides a method for screeningan agent which modulates cancer metastasis or cancer cell migrationcomprising:

(a) contacting a testing agent with KRS in the presence of the testingagent;

(b) measuring activity of KRS and selecting a testing agent whichchanges activity of KRS; and

(c) testing whether the selected agent regulates tumor metastasis orcancer cell migration.

In another aspect, the present invention provides a method for screeningan agent inhibiting an interaction between KRS and 67LR comprising:

(a) contacting a testing agent with KRS and laminin receptor (67LR) inthe presence of the testing agent; and

(b) testing whether the selected agent regulates an interaction betweenKRS and laminin receptor.

In another aspect, the present invention provides a method for diagnosisof lung cancer or breast cancer comprising:

(a) analyzing overexpression of 67LR in a sample; and

(b) analyzing overexpression of KRS in the 67LR over-expressed sample.

Hereinafter, the present invention will be described in detail.

In the present invention, the present inventors first identified thatKRS has an effect on cancer metastasis or cancer cell migration. Thatis, the present inventor identified that KRS has an effect on cancermetastasis or cancer cell migration through a laminin receptor in theplasma membrane.

DEFINITION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOTY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); and Hale & Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY. In addition, the following definitions areprovided to assist the reader in the practice of the invention.

An “expression”, as used herein, refers to formation of protein ornucleic acid in cells.

A “host cell,” as used herein, refers to a prokaryotic or eukaryoticcell that contains heterologous DNA that has been introduced into thecell by any means, e.g., electroporation, calcium phosphateprecipitation, microinjection, transformation, viral infection, and/orthe like.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring nucleic acid,polypeptide, or cell present in a living animal is not isolated, but thesame polynucleotide, polypeptide, or cell separated from some or all ofthe coexisting materials in the natural system, is isolated, even ifsubsequently reintroduced into the natural system. Such nucleic acidscan be part of a vector and/or such nucleic acids or polypeptides couldbe part of a composition, and still be isolated in that such vector orcomposition is not part of its natural environment.

The term “modulate” with respect to KRS bioactivities refers to a changein the cellular level of KRS. Modulation of KRS bioactivities can beup-regulation (i.e., activation or stimulation) or down-regulation (i.e.inhibition or suppression). For example, modulation may cause a changein cellular level of KRS, stability of protein, enzymatic modification(e.g., phosphorylation) of KRS, binding characteristics (e.g., bindingto a target transcription regulatory element), or any other biological,functional, or immunological properties of KRS. The change in activitycan arise from, for example, an increase or decrease in expression ofthe KRS gene, the stability of mRNA that encodes the KRS protein,translation efficiency, or from a change in other bioactivities of theKRS transcription factor (e.g., regulating expression of aKRS-responsive gene). The mode of action of a KRS modulator can bedirect, e.g., through binding to the KRS protein or to genes encodingthe KRS protein. The change can also be indirect, e.g., through bindingto and/or modifying (e.g., enzymatically) another molecule whichotherwise modulates KRS (e.g., a kinase that specifically phosphorylatesKRS).

The term “polypeptide” is used interchangeably herein with the terms“polypeptides” and “protein(s)”, and refers to a polymer of amino acidresidues, e.g., as typically found in proteins in nature.

The term “KRS polypeptide,” refers to a polypeptide known as lysyl tRNAsynthetase. The said KRS polypeptide may be a polypeptide having anamino acid sequence of SEQ ID NO: 1 (GenBank Accession No: NP_005539.1).And the inventive KRS includes functional equivalents thereof.

The term “functional equivalents” refers to polypeptide comprising theamino acid sequence having at least 70% amino acid sequence homology(i.e., identity), preferably at least 80%, and more preferably at least90%, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence homology, thatexhibit substantially identical physiological activity to thepolypeptide of SEQ ID NO: 1. The “substantially identical physiologicalactivity” means interaction with laminin receptor of plasma membrane andregulation of tumor metastasis or tumor cell migration. The functionalequivalents may include, for example peptides produced by as a result ofaddition, substitution or deletion of some amino acid of SEQ ID NO:1.Substitutions of the amino acids are preferably conservativesubstitutions. Examples of conservative substitutions of naturallyoccurring amino acids are as follows: aliphatic amino acids (Gly, Ala,Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids(Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His,Lys, Arg, Gln, Asn) and sulfur-containing amino acids (Cys, Met).Furthermore, the functional equivalents also include variants withdeletion of some of the amino acid sequence of the inventive KRS.Deletion or substitutions of the amino acids are preferably located atregions that are not directly involved in the physiological activity ofthe inventive polypeptide. And deletion of the amino acids is preferablylocated at regions that are not directly involved in the physiologicalactivity of the KRS. In addition, the functional equivalents alsoinclude variants with addition of several amino acids in both terminalends of the amino acid sequence of the KRS or in the sequence. Moreover,the inventive functional equivalents also include polypeptidederivatives which have modification of some of the chemical structure ofthe inventive polypeptide while maintaining the fundamental backbone andphysiological activity of the inventive polypeptide. Examples of thismodification include structural modifications for changing thestability, storage, volatility or solubility of the inventivepolypeptide.

Sequence identity or homology is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withamino acid sequence of KRS (SEQ ID NO: 1), after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions(as described above) as part of the sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the amino acid sequence of KRS shall be construed as affectingsequence identity or homology. Thus, sequence identity can be determinedby standard methods that are commonly used to compare the similarity inposition of the amino acids of two polypeptides. Using a computerprogram such as BLAST or FASTA, two polypeptides are aligned for optimalmatching of their respective amino acids (either along the full lengthof one or both sequences or along a predetermined portion of one or bothsequences). The programs provide a default opening penalty and a defaultgap penalty, and a scoring matrix such as PAM 250 (a standard scoringmatrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure,vol. 5, supp. 3 (1978)) can be used in conjunction with the computerprogram. For example, the percent identity can be calculated as thefollow. The total number of identical matches multiplied by 100 and thendivided by the sum of the length of the longer sequence within thematched span and the number of gaps introduced into the longer sequencesin order to align the two sequences.

The polypeptide according to the present invention can be prepared byseparating from nature materials or genetic engineering methods. Forexample, a DNA molecule encoding the KRS or its functional equivalents(ex: SEQ ID NO: 2 (Genbank Accession No. D32053)) is constructedaccording to any conventional method. The DNA molecule may synthesize byperforming PCR using suitable primers. Alternatively, the DNA moleculemay also be synthesized by a standard method known in the art, forexample using an automatic DNA synthesizer (commercially available fromBiosearch or Applied Biosystems). The constructed DNA molecule isinserted into a vector comprising at least one expression controlsequence (ex: promoter, enhancer) that is operatively linked to the DNAsequence so as to control the expression of the DNA molecule, and hostcells are transformed with the resulting recombinant expression vector.The transformed cells are cultured in a medium and condition suitable toexpress the DNA sequence, and a substantially pure polypeptide encodedby the DNA sequence is collected from the culture medium. The collectionof the pure polypeptide may be performed using a method known in theart, for example, chromatography. In this regard, the term“substantially pure polypeptide” means the inventive polypeptide thatdoes not substantially contain any other proteins derived from hostcells. For the genetic engineering method for synthesizing the inventivepolypeptide, the reader may refer to the following literatures. Maniatiset al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory 1982; Sambrook et al., Molecular Cloning; A LaboratoryManual, Cold Spring Harbor Press, N.Y., Second (1998) and Third (2000)Editions; Gene Expression Technology, Method in Enzymology, Genetics andMolecular Biology, Method in Enzymology, Guthrie & Fink (eds.), AcademicPress, San Diego, Calif. 1991; and Hitzeman et al., J. Biol. Chem., 255,12073-12080 1990.

Alternatively, the inventive polypeptide can be chemically synthesizedeasily according to any technique known in the art (Creighton, Proteins:Structures and Molecular Principles, W.H. Freeman and Co., NY, 1983). Asa typical technique, they are not limited to, but include liquid orsolid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry(Chemical Approaches to the Synthesis of Peptides and Proteins, Williamset al., Eds., CRC Press, Boca Raton Fla., 1997; A Practical Approach,Atherton & Sheppard, Eds., IRL Press, Oxford, England, 1989).

The inventive laminin receptor (67LR) of 67 kDa is plasmamembrane-embeded, non-integrin receptor and for example, it may have anucleotide sequence or amino acid sequence any one disclosed in GenbankAccession No. NM_002295, S37431, AF284768, S37431, AF284768, J03799, XP370865, XP 001083023.

The terms “nucleic acid,” “DNA sequence” or “polynucleotide” refer to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues of natural nucleotides that hybridize to nucleic acids inmanner similar to naturally occurring nucleotides.

The term “the nucleotide encoding KRS or functional equivalents thereof”may have a nucleic acid encoding a polypeptide having the amino acidsequence of SEQ ID NO: 1 or a polypeptide having the amino acid sequencehomology of at least 70% to the polypeptide. The nucleic acid includesDNA, cDNA or RNA. The polynucleotide may have a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 1 or an amino acidsequence homology of at least 70% to SEQ ID NO: 1. Preferably, thepolynucleotide comprises the nucleotide sequence of SEQ ID NO. 2. Thenucleic acid can be isolated from a natural source or be prepared by agenetic engineering method known in the art.

The term “analog” is used herein to refer to a molecule thatstructurally resembles a reference molecule but which has been modifiedin a targeted and controlled manner, by replacing a specific substituentof the reference molecule with an alternate substituent. Compared to thereference molecule, an analog would be expected, by one skilled in theart, to exhibit the same, similar, or improved utility. Synthesis andscreening of analogs, to identify variants of known compounds havingimproved traits (such as higher binding affinity for a target molecule)is an approach that is well known in pharmaceutical chemistry.

The term “homologous” when referring to proteins and/or proteinsequences indicates that they are derived, naturally or artificially,from a common ancestral protein or protein sequence. Similarly, nucleicacids and/or nucleic acid sequences are homologous when they arederived, naturally or artificially, from a common ancestral nucleic acidor nucleic acid sequence.

As used herein, the term “effective amount” refers to an amount showingan effect of the modulating KRS bioactiviy (ex: cellular levels etc.)differently to normal cells or tissues or the inhibiting theubiquitination of KRS.

As used herein, “contacting” has its normal meaning and refers tocombining two or more agents (e.g., two polypeptides) or combiningagents and cells (e.g., a protein and a cell). Contacting can occur invitro, e.g., combining two or more agents or combining a test agent anda cell or a cell lysate in a test tube or other container. Contactingcan also occur in a cell or in situ, e.g., contacting two polypeptidesin a cell by coexpression in the cell of recombinant polynucleotidesencoding the two polypeptides, or in a cell lysate.

The term “agent” or “test agent” includes any substance, molecule,element, compound, entity, or a combination thereof. It includes, but isnot limited to, e.g., protein, polypeptide, small organic molecule,polysaccharide, polynucleotide, and the like. It can be a naturalproduct, a synthetic compound, or a chemical compound, or a combinationof two or more substances. Unless otherwise specified, the terms“agent”, “substance”, and “compound” can be used interchangeably.

More specifically, test agents that can be identified with methods ofthe present invention include polypeptides, beta-turn mimetics,polysaccharides, phospholipids, hormones, prostaglandins, steroids,aromatic compounds, heterocyclic compounds, benzodiazepines, oligomericN-substituted glycines, oligocarbamates, polypeptides, saccharides,fatty acids, steroids, purines, pyrimidines, derivatives, structuralanalogs or combinations thereof. Some test agents are syntheticmolecules, and others natural molecules. Test agents are obtained from awide variety of sources including libraries of synthetic or naturalcompounds. Combinatorial libraries can be produced for many types ofcompound that can be synthesized in a step-by-step fashion. Largecombinatorial libraries of compounds can be constructed by the encodedsynthetic libraries (ESL) method described in WO 95/12608, WO 93/06121,WO 94/08051, WO 95/35503 and WO 95/30642. Peptide libraries can also begenerated by phage display methods (see, e.g., Devlin, WO 91/18980).Libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts can be obtained from commercial sources or collectedin the field. Known pharmacological agents can be subject to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification to produce structural analogs.

The test agents can be naturally occurring proteins or their fragments.Such test agents can be obtained from a natural source, e.g., a cell ortissue lysate. Libraries of polypeptide agents can also be prepared,e.g., from a cDNA library commercially available or generated withroutine methods. The test agents can also be peptides, e.g., peptides offrom about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred and from about 7 to about 15 beingparticularly preferred. The peptides can be digests of naturallyoccurring proteins, random peptides, or “biased” random peptides.

The test agents can also be “nucleic acids”. Nucleic acid test agentscan be naturally occurring nucleic acids, random nucleic acids, or“biased” random nucleic acids. For example, digests of prokaryotic oreukaryotic genomes can be similarly used as described above forproteins.

In some preferred methods, the test agents are small molecules (e.g.,molecules with a molecular weight of not more than about 1,000).Preferably, high throughput assays are adapted and used to screen forsuch small molecules. A number of assays are available for suchscreening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett8:2409-2414; The present inventorsller (1997) Mol Divers. 3:61-70;Fernandes (1998) Curr Opin Chem Biol 2:597-603; and Sittampalam (1997)Curr Opin Chem Biol 1:384-91.

Libraries of test agents to be screened with methods of the presentinvention can also be generated based on structural studies of the KRS,its fragment or its analog. Such structural studies allow theidentification of test agents that are more likely to bind to the KRS.The three-dimensional structures of the KRS can be studied in a numberof ways, e.g., crystal structure and molecular modeling. Methods ofstudying protein structures using x-ray crystallography are well knownin the literature. See Physical Bio-chemistry, Van Holde, K. E.(Prentice-Hall, New Jersey 1971), pp. 221-239, and Physical Chemistrywith Applications to the Life Sciences, D. Eisenberg & D. C. Crothers(Benjamin Cummings, Menlo Park 1979). Computer modeling of structures ofKRS provides another means for designing test agents for screening KRS.Methods of molecular modeling have been described in the literature,e.g., U.S. Pat. No. 5,612,894 entitled “System and method for molecularmodeling utilizing a sensitivity factor”, and U.S. Pat. No. 5,583,973entitled “Molecular modeling method and system”. In addition, proteinstructures can also be determined by neutron diffraction and nuclearmagnetic resonance (NMR). See, e.g., Physical Chemistry, 4th Ed. Moore,W. J. (Prentice-Hall, New Jersey 1972), and NMR of Proteins and NucleicAcids, K. Wuthrich (Wiley-Interrscience, New York 1986).

Hereinafter, the present invention will be described in detail.

The present inventors disclosed that the inventive KRS interacts with67LR through translocation of KRS into plasma membrane, and so enhancestumor (or cancer) cell migration, thereby having an effect on cancermetastasis. In addition, we also disclosed that KRS overexpression orinhibition of KRS expression can modulate tumor (or cancer) cellmetastasis through in vivo experiments using mice.

Accordingly, the present invention provides a method for controllingcancer metastasis by modulating a cellular level of lysyl tRNAsynthetase.

To be explained more in detail, if the cellular level of the inventivelysyl tRNA synthetase is reduced, the cancer metastasis may besuppressed, and if the cellular level of the inventive lysyl tRNAsynthetase is increased, the cancer metastasis may be stimulated.

The reduction or increase of the cellular level is regulated withvarious well known methods in the art as described above. For example,but not limited thereto, the cellular level may be controlled throughtranscriptional regulation or post-transcriptional regulation. Thetranscriptional regulation may be performed by the method of enhancing agene expression known in the art, e.g., the method of enhancing a geneexpression by preparing a recombinant expression vector comprising apolynucleotide encoding KRS or functional equivalent thereof operablylinked to a promoter, or the method for inserting an expressionregulating sequence to enhance an expression of a gene encoding KRS orfunctional equivalent thereof around the gene, or the method forinhibiting gene expression, e.g., the method inhibiting promoteractivity or protein function by inducing mutation in promoter or generegion, the method for expressing antisense gene, or siRNA or microRNA.

The post-transcriptional regulation may be performed by the method forenhancing or suppressing protein expression known in the art, e.g., themethod for enhancing or suppressing stability of mRNA of the geneencoding KRS or functional equivalent thereof, the method for enhancingor suppressing stability of the protein or the polypeptide, or themethod for enhancing or suppressing activity of the protein or thepolypeptide.

For concrete example of the above mentioned methods, it can inducecosuppression via transformation using DNA sequence encoding RNA actingto mRNA such as type 1 intron, M1 RNA type, hammerhead type or hairpintype or micro RNA type, or transformation using DNA having the same orsimilar sequence to a target gene.

Preferably, in the present invention, the method for controlling of thecellular level of KRS or a functional equivalent thereof may beperformed by the method for enhancing or suppressing expression of apolynucleotide encoding the polypeptide. The method for enhancing orsuppressing may be used by skilled persons in the art, for example,through preparing of a recombinant expression vector comprising apolynucleotide encoding KRS or a functional equivalent thereof operablylinked to a promoter to enhance the expression of the polynucleotide, orthrough preparing of a recombinant expression vector comprisingantisense RNA polynucleotide or siRNA polynucleotide against thepolynucleotide encoding KRS or a functional equivalent thereof operablylinked to a promoter to suppress the expression of the polynucleotide.The polynucleotide encoding KRS or a functional equivalent thereof mayhave the nucleotide sequence represented by SEQ ID.NO:2, preferably.

In addition, the present invention provides a method for controllingcancer cell migration by modulating a cellular level of lysyl tRNAsynthetase, and the modulation the cellular level is as same asdescribed above.

In addition, when the expression of the inventive KRS is suppressed,tumor (or cancer) metastasis is inhibited, the present inventionprovides a composition for preventing and treating cancer comprising anexpression vector comprising a promoter and a structural genesuppressing expression of KRS operably linked thereto, or an antibodyagainst KRS as an effective ingredient. The structural gene suppressingexpression of KRS may be antisense RNA or siRNA against a polynucleotideencoding KRS.

The diseases which can be applied the inventive composition may becancers. The cancers include, but are not limited to, colon cancer, lungcancer, liver cancer, stomach cancer, esophagus cancer, pancreaticcancer, gall bladder cancer, kidney cancer, bladder cancer, prostatecancer, testis cancer, cervical cancer, endometrial carcinoma,choriocarcinoma, ovarian cancer, breast cancer, thyroid cancer, braintumor, head or neck cancer, malignant melanoma, lymphoma, aplasticanemia.

The “promoter” means a DNA sequence regulating the expression of nucleicacid sequence operably linked to the promoter in a specific host cell,and the term “operably linked” means that one nucleic acid fragment islinked to other nucleic acid fragment so that the function or expressionthereof is affected by the other nucleic acid fragment. Additionally,the promoter may include an operator sequence for controllingtranscription, a sequence encoding a suitable mRNA ribosome-bindingsite, and sequences controlling the termination transcription andtranslation. Additionally, it may be constitutive promoter whichconstitutively induces the expression of a target gene, or induciblepromoter which induces the expression of a target gene at a specificsite and a specific time, and examples thereof include a SV40 promoter,CMV promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108:193-199,1991; Monahan et al., Gene Therapy, 7:24-30, 2000), CaMV 35S promoter(Odell et al., Nature 313:810-812, 1985), Rsyn7 promoter (U.S. patentapplication Ser. No. 08/991,601), rice actin promoter (McElroy et al.,Plant Cell 2:163-171, 1990), Ubiquitin promoter (Christensen et al.,Plant Mol. Biol. 12:619-632, 1989), ALS promoter (U.S. patentapplication Ser. No. 08/409,297). Also usable promoters are disclosed inU.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; and 5,608,142, etc.)

Meanwhile, the present invention provides a method for preventing andtreating cancer comprising administering to a subject in need thereof aneffective amount of an expression vector comprising a promoter and astructural gene suppressing expression of KRS operably linked thereto,or an antibody against KRS. As the structure gene is described above,the present invention provides a method for preventing and treatingcancer comprising administering to a subject in need thereof aneffective amount of an expression vector comprising a promoter and apolynucleotide operably linked thereto, or an antibody against KRS as aneffective ingredient, wherein the polynucleotide is encoding antisenseRNA or siRNA against the KRS polynucleotide.

As used herein, the “effective amount” refers to the amount of inventiveexpression vector effective in treating tumor, and the “subject” refersto animals, preferably, animals comprising human and it may be cells,tissues, organs originated from the animals. The subject may be patientin need of treatment.

In addition, the present invention provides an use of an expressionvector comprising a promoter and a structural gene suppressingexpression of KRS operably linked thereto, or an antibody against KRSfor preparation an anti-cancer agent. More specifically, the presentinvention provides an use of an expression vector comprising a promoterand a polynucleotide operably linked thereto, or an antibody against KRSfor preparation an anti-cancer agent, wherein the polynucleotide isencoding antisense RNA or siRNA against the KRS polynucleotide.Regarding the above promoter, KRS, expression vector, applied cancersare as can be seen from the foregoing.

The term antibody against KRS as used herein means a specific proteinmolecule that indicates an antigenic region concerning antigenic regionof KRS. With respect to the objects of the present invention, theantibody refers to an antibody specifically binding KRS and includes allpolyclonal, monoclonal and recombinant antibodies.

The antibodies against the KRS may be easily prepared in accordance withconventional technologies known to one skilled in the art. Polyclonalantibodies may be prepared by a method widely known in the art, whichincludes injecting the KRS protein into an animal and collecting bloodsamples from the animal to obtain serum containing antibodies. Suchpolyclonal antibodies may be prepared from a certain animal host, suchas goats, rabbits, sheep, monkeys, horses, pigs, cows and dogs.

Monoclonal antibodies may be prepared by a method widely known in theart, such as a hybridoma method (Kohler and Milstein, European Journalof Immunology, 6:511-519(1976)) or a phage antibody library technique(Clackson et al, Nature, 352:624-628(1991); and Marks et al, J. Mol.Biol., 222:58, 1-597(1991)).

The hybridoma method employs cells from an immunologically suitable hostanimal injected with a diagnostic marker protein of lung cancer as anantigen, such as mice, and a cancer or myeloma cell line as anothergroup. Cells of the two groups are fused with each other by a methodwidely known in the art, for example, using polyethylene glycol, andantibody-producing cells are proliferated by a standard tissue culturemethod. After uniform cell colonies are obtained by subcloning using alimited dilution technique, hybridomas capable of producing an antibodyspecific for the diagnostic marker protein of lung cancer are cultivatedin large scale in vitro or in vivo according to a standard technique.Monoclonal antibodies produced by the hybridomas may be used in anunpurified form, but are preferably used after being highly purified bya method widely known in the art so as to obtain best results. The phageantibody library method includes constructing a phage antibody libraryin vitro by obtaining genes for antibodies (single-chain fragmentvariable (scFv)) to a variety of intracellular lung cancer markers andexpressing them in a fusion protein form on the surface of phages, andisolating monoclonal antibodies binding to lung cancer-specific proteinsfrom the library. Antibodies prepared by the above methods are isolatedusing gel electrophoresis, dialysis, salting out, ion exchangechromatography, affinity chromatography, and the like.

In addition, the antibodies of the present invention include completeforms having two full-length light chains and two full-length heavychains, as well as functional fragments of antibody molecules. Thefunctional fragments of antibody molecules refer to fragments retainingat least an antigen-binding function, and include Fab, F(ab′), F(ab′)2and Fv.

In addition, so the present inventor disclosed that in case ofdecreasing the cellular level of KRS, it suppress cancer metastasis,thereby preventing and treating cancer, and the present inventionprovides a composition for preventing and treating cancer comprising aKRS activity inhibitor as an effective ingredient. In addition, thepresent invention provides a method for preventing and treating cancercomprising administering to a subject in need thereof effective amountof a KRS activity inhibitor and a use of a KRS activity inhibitor forpreparing anti-cancer agent. The type of cancer, the subject, effectiveamount and so on are the same as described above.

The KRS activity inhibitor means the agent for suppressing expression ofKRS, that is, suppressing expression in the level of mRNA or protein,for example, it may be antisense RNA or siRNA against KRS, orcompetitive inhibitor or non-competitive inhibitor for suppressingactivity of expressed KRS, for example, antibodies against KRS, but notlimited thereto.

In case of decreasing the cellular level of KRS, since it inhibitscancer metastasis to prevent and treat cancer, the inventivecomposition, method and use may be applied as themselves as well as maybe applied as combinations with well known method for preventing andtreating cancer in the art. That is, since the inventive composition,method and etc. can suppress cancer metastasis, if it is appliedtogether with well known anticancer drugs or methods for preventing andtreating cancer, it suppresses cancer metastasis and would be effectivefor full recovery through treatment of main tumor region.

The antitumor agent or the method for preventing and treating that canbe used in combination with the polypeptide of the present invention maybe any one that is used for treatment of a tumor. For example,paclitaxel, doxorubicin, vincristine, daunorubicin, vinblastine,daunorubicin D, docetaxel, etoposide, teniposide, bisantrene,homoharringtonine, Gleevec (STI-571), cisplatin, 5-fluorouracil,Adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide,melphalan, nitrogen mustard, nitrosourea, etc. may be included. Theamount of the peptide of the present invention included in thecomposition of the present invention may be different depending on thekind and amount of the anticancer drug that the peptide binds to.

The combinations between the agents, and the composition or the methodof the present invention may be performed depending on the kind and theamount of the anticancer drug appropriately by the skilled person in theart.

The inventive expression vector, the agent inhibiting activities ofantibody against KRS or KRS may be administered orally or parenterally.The oral administration may comprise hypoglossal method. Parenteraladministration methods are not limited, but include injection methodssuch as hypodermical, intramuscular and intravenous, and droppingmethod. The inventive expression vector, the agent inhibiting activitiesof antibody against KRS or KRS may be prepared into various types ofpharmaceutical compositions by mixing with pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutically acceptable” meanswhat is physiologically acceptable and, when administered to humanbeings, generally does not cause allergic reactions, such asgastrointestinal disorder and dizziness, or similar reactions thereto.In the case of an oral formulation, a binding agent, a lublitant, asolutionizer, an exipient, a solubilizer, a dispersing agent, astabilizer, a suspending agent, a colorant and a flavor may be used. Inthe case of an injection formulation, a buffer, a preservative, apainless agent, a solubilizer, a isotonic agent and a stabilizer may beused and In the case of an or parenteral formulation, a base, anexipient, a lubricant and a preservative may be used. The pharmaceuticalcomposition comprising the inventive expression vector, the agentinhibiting activities of antibody against KRS or KRS may be preparedinto various types by mixing with pharmaceutically acceptable carriers.For example, in the case of an oral formulation, it may be formulatedinto tablet, troche, capsule, elixir, suspension, syrup, and wafer andin the case of injection formulation; it may be prepared into a singledose ampoule or a multiple dose ampoule.

The total effective amount of the inventive expression vector, the agentinhibiting activities of antibody against KRS or KRS can be administeredto a subject as a single dose, or can be administered using afractionated treatment protocol, in which the multiple doses areadministered over a more prolonged period of time. The compositioncomprising the inventive expression vector, the agent inhibitingactivities of antibody against KRS or KRS can be varied in amount ofeffective component depending on the severity and/or object of disease,but normally, it may be administered by 0.1 μg to 100 mg, and preferably1 μg to 10 mg and multiple times a day. However, one skilled in the artwould know that the concentration of the inventive expression vector orthe agent inhibiting activities of KRS required to obtain an effectivedose in a subject depends on many factors including the age, bodyweight, health condition, disease severity, diet and excretion of thesubject, the route of administration, the number of treatments to beadministered, and so forth. In view of these factors, any person skilledin the art can determine the suitable effective dose of the inventiveexpression vector or the agent inhibiting activities of KRS. Noparticular limitation is imposed on the formulation, administrationroute and administration mode of the pharmaceutical compositionaccording to the present invention, as long as the composition shows theeffects of the present invention.

The inventive compositions may be administered to patients with theamount which is effective for preventing disease. Generally, theeffective amount of the inventive composition is about 0.0001 to 100mg/kg body weight/day. Preferably 0.01 to 1 mg/kg body the presentweight/day. It may be suitably determined by considering variousfactors, such as age, body weight, health condition, sex, diseaseseverity, diet and excretion of a subject in need of treatment, as wellas administration time and administration route.

Meanwhile, the said expression vector can be introduced into a targetcell by any method known in the art, such as infection, transfection ortransduction.

A gene transfer method using a plasmid expression vector is a method oftransferring a plasmid DNA directly to mammalian cells, which is anFDA-approved method applicable to human beings (Nabel, E. G., et al.,Science, 249:1285-1288, 1990). Unlike viral vectors, the plasmid DNA hasan advantage of being homogeneously purified. Plasmid expression vectorswhich can be used in the present invention include mammalian expressionplasmids known in the pertinent art. For example, they are not limitedto, but typically include pRKS (European Patent No. 307,247), pSV16B(PCT Publication No. 91/08291) and pVL1392 (PharMingen). The plasmidexpression vector containing the said polynucleotide may be introducedinto target cells by any method known in the art, including, but notlimited to, transient transfection, microinjection, transduction, cellfusion, calcium phosphate precipitation, liposome-mediated transfection,DEAE dextran-mediated transfection, polybrene-mediated transfection,electroporation, gene gun methods, and other known methods forintroducing DNA into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992;Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).

In addition, virus expression vectors containing the said polynucleotideinclude, but are not limited to, retrovirus, adenovirus, herpes virus,avipox virus and so on. The retroviral vector is so constructed thatnon-viral proteins can be produced within the infected cells by theviral vector in which virus genes are all removed or modified. The mainadvantages of the retroviral vector for gene therapy are that ittransfers a large amount of genes into replicative cells, preciselyintegrates the transferred genes into cellular DNA, and does not inducecontinuous infections after gene transfection (Miller, A. D., Nature,357:455-460, 1992). The retroviral vector approved by FDA was preparedusing PA317 amphotropic retrovirus packaging cells (Miller, A. D. andButtimore, C., Molec. Cell Biol., 6:2895-2902, 1986). Non-retroviralvectors include adenovirus as described above (Rosenfeld et al., Cell,68:143-155, 1992; Jaffe et al., Nature Genetics, 1:372-378, 1992;Lemarchand et al., Proc. Natl. Acad. Sci. USA, 89:6482-6486, 1992). Themain advantages of adenovirus are that it transfers a large amount ofDNA fragments (36 kb genomes) and is capable of infectingnon-replicative cells at a very high titer. Moreover, herpes virus mayalso be useful for human genetic therapy (Wolfe, J. H., et al., NatureGenetics, 1:379-384, 1992). Besides, other known suitable viral vectorscan be used.

In addition, a vector capable of inhibiting expressing the expression ofKRS may be administered by a known method. For example, the vector maybe administered locally, parenterally, orally, intranasally,intravenously, intramuscularly or subcutaneously, or by other suitableroutes. Particularly, the vector may be injected directly into a targetcancer or tumor cell at an effective amount for treating the tumor cellof a target tissue. Particularly for a cancer or tumor present in a bodycavity such as in the eye, gastrointestinal tract, genitourinary tract,pulmonary and bronchial system and so on, the inventive pharmaceuticalcomposition can be injected directly into the hollow organ affected bythe cancer or tumor using a needle, a catheter or other delivery tubes.Any effective imaging device, such as X-ray, sonogram, or fiberopticvisualization system, may be used to locate the target tissue and guidethe needle or catheter tube. In addition, the inventive pharmaceuticalcomposition may be administered into the blood circulation system fortreatment of a cancer or tumor which cannot be directly reached oranatomically isolated.

The present invention also provides a method for screening an agentwhich modulates cancer metastasis or cancer cell migration comprising:

(a) contacting a testing agent with KRS in the presence of the testingagent;

(b) measuring activity of KRS and selecting a testing agent whichchanges activity of KRS; and

(c) testing whether the selected agent regulates tumor metastasis orcancer cell migration

Various biochemical and molecular biology techniques or assays wellknown in the art can be employed to practice the present invention. Suchtechniques are described in, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, N.Y., Second (1989) andThird (2000) Editions; and Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., New York (1987-1999).

Preferably, the test agent is first assayed for their ability tomodulate a biological activity of KRS (the first assay step).Particularly, in the first step, modulating agents that modulate abiological activity of an the said polypeptide may be identified byassaying a biological activity of isolated KRS in the presence of a testagent. More preferably, the present invention may comprise:

(a) contacting test agents with KRS in the presence of a test agent; and

(b) measuring activity of KRS and selecting a testing agent whichchanges activity of KRS.

Modulation of different biological activities of KRS can be assayed inthe first step. For example, a test agent can be assayed for activity tomodulate expression level of KRS, e.g., transcription or translation.The test agent can also be assayed for activities in modulating cellularlevel or stability of KRS, e.g., post-translational modification orproteolysis.

Test agents that increase a biological activity of KRS by the firstassay step are identified, the test agents are then subject to furthertesting for ability to modulate an activity of laminin receptor (67LR),in the presence of KRS (the second testing step). For example, the testagents are then subject to further testing for ability to modulatecancer metastasis or tumor cell migration.

As noted above, the KRS-modulating agents identified by the presentinvention can modulate cancer metastasis or tumor cell migration. If atest agent identified in the first testing step modulates cellular level(e.g., by altering transcription activity) of the KRS-modulating agents,it would modulate cancer metastasis or tumor cell migration.

On the other hand, if a test agent modulates an activity other thancellular level of KRS, then the further testing step is needed toconfirm that their modulatory effect on the KRS would indeed lead tomodulation of cancer metastasis or tumor cell migration. For example, atest agent, which modulates phosphorylation activity of KRS, needs to befurther tested in order to confirm that modulation of phosphorylationactivity of KRS can result in modulation of cancer metastasis or tumorcell migration.

In both the first step and the second step, an intact KRS and subunitsor their fragments, analogs, or functional derivatives can be used. Thefragments that can be employed in these assays usually retain one ormore of the biological activities of KRS. Preferably, AIMP2 fragmentsmay comprise 1^(st)-72^(nd) amino acid residues of SEQ. ID NO: 1. Andfusion proteins containing such fragments or analogs can also be usedfor the screening of test agents. Functional derivatives of KRS usuallyhave amino acid deletions and/or insertions and/or substitutions whilemaintaining one or more of the bioactivities and therefore can also beused in practicing the screening methods of the present invention.

A variety of the well-known techniques can be used to identify testagents that modulate KRS. Preferably, the test agents are screened witha cell based assay system. For example, in a typical cell based assay(i.e., the second screening step), activity of the reporter gene (i.e.,enzyme activity) is measured in the presence of test agent, and thencompared the activity of the reporter gene in the absence of test agent.The reporter gene can encode any detectable polypeptide (response orreporter polypeptide) known in the art, e.g., detectable by fluorescenceor phosphorescence or by virtue of its possessing an enzymatic activity.The detectable response polypeptide can be, e.g., luciferase,alpha-glucuronidase, alpha-galactosidase, chloramphenicol acetyltransferase, green fluorescent protein, enhanced green fluorescentprotein, and the human secreted alkaline phosphatase.

In the cell-based assays, the test agent (e.g., a peptide or apolypeptide) can also be expressed from a different vector that is alsopresent in the host cell. In some methods, a library of test agents isencoded by a library of such vectors (e.g., a cDNA library; see theExample below). Such libraries can be generated using methods well knownin the art (see, e.g., Sambrook et al. and Ausubel et al., supra) orobtained from a variety of commercial sources.

In addition to cell based assays described above, it can also bescreened with non-cell based methods. These methods include, e.g.,mobility shift DNA-binding assays, methylation and uracil interferenceassays, DNase and hydroxy radical footprinting analysis, fluorescencepolarization, and UV crosslinking or chemical cross-linkers. For ageneral overview, see, e.g., Ausubel et al., supra (chapter 12,DNA-Protein Interactions). One technique for isolating co-associatingproteins, including nucleic acid and DNA/RNA binding proteins, includesuse of UV crosslinking or chemical cross-linkers, including e.g.,cleavable cross-linkers dithiobis (succinimidylpropionate) and3,3′-dithiobis (sulfosuccinimidyl-propionate); see, e.g., McLaughlin,Am. J. Hum. Genet., 59:561-569, 1996; Tang, Biochemistry, 35:8216-8225,1996; Lingner, Proc. Natl. Acad. Sci. U.S.A., 93:10712, 1996; andChodosh, Mol. Cell. Biol., 6:4723-4733, 1986.

First Assay Step: Screening Test Agents that Modulate KRS

A number of assay systems can be employed to screen test agents formodulators of KRS. As noted above, the screening can utilize an in vitroassay system or a cell-based assay system. In this screening step, testagents can be screened for binding to KRS, altering cellular level ofKRS, or modulating other biological activities of KRS.

1) Screening of Test Agents that Bind KRS

In the first screening step some methods, binding of a test agent to KRSis determined. For example, it can be assayed by a number of methodsincluding e.g., labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.), and the like. See,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168; andalso Bevan et al., Trends in Biotechnology 13:115-122, 1995; Ecker etal., Bio/Technology 13:351-360, 1995; and Hodgson, Bio/Technology10:973-980, 1992. The test agent can be identified by detecting a directbinding to KRS, e.g., co-immunoprecipitation with KRS by an antibodydirected to KRS. The test agent can also be identified by detecting asignal that indicates that the agent binds to KRS, e.g., fluorescencequenching.

Competition assays provide a suitable format for identifying test agentsthat specifically bind to KRS. In such formats, test agents are screenedin competition with a compound already known to bind to KRS. The knownbinding compound can be a synthetic compound. It can also be anantibody, which specifically recognizes KRS polypeptide, e.g., amonoclonal antibody directed against KRS. If the test agent inhibitsbinding of the compound known to bind KRS, then the test agent alsobinds KRS.

Numerous types of competitive binding assays are known, for example:solid phase direct or indirect radioimmunoassay (RIA), solid phasedirect or indirect enzyme immunoassay (EIA), sandwich competition assay(see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phasedirect biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA using.sup.125I label (see Morel et al., Mol. Immunol. 25(1):7-15 (1988));solid phase direct biotin-avidin EIA (Cheung et al., Virology176:546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand.J. Immunol. 32:77-82 (1990)). Typically, such an assay involves the useof purified polypeptide bound to a solid surface or cells bearing eitherof these, an unlabelled test agent and a labeled reference compound.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the test agent.Usually the test agent is present in excess. Modulating agentsidentified by competition assay include agents binding to the sameepitope as the reference compound and agents binding to an adjacentepitope sufficiently proximal to the epitope bound by the referencecompound for steric hindrance to occur. Usually, when a competing agentis present in excess, it will inhibit specific binding of a referencecompound to a common target polypeptide by at least 50 or 75%.

The screening assays can be either in insoluble or soluble formats. Oneexample of the insoluble assays is to immobilize KRS or its fragmentsonto a solid phase matrix. The solid phase matrix is then put in contactwith test agents, for an interval sufficient to allow the test agents tobind. Following washing away any unbound material from the solid phasematrix, the presence of the agent bound to the solid phase allowsidentification of the agent. The methods can further include the step ofeluting the bound agent from the solid phase matrix, thereby isolatingthe agent. Alternatively, other than immobilizing KRS, the test agentsare bound to the solid matrix and the KRS is then added.

Soluble assays include some of the combinatory libraries screeningmethods described above. Under the soluble assay formats, neither thetest agents nor KRS are bound to a solid support. Binding of KRS orfragment thereof to a test agent can be determined by, e.g., changes influorescence of either KRS or the test agents, or both. Fluorescence maybe intrinsic or conferred by labeling either component with afluorophor.

In some binding assays, either KRS, the test agent, or a third molecule(e.g., an antibody against KRS) can be provided as labeled entities,i.e., covalently attached or linked to a detectable label or group, orcross-linkable group, to facilitate identification, detection andquantification of the polypeptide in a given situation. These detectablegroups can comprise a detectable polypeptide group, e.g., an assayableenzyme or antibody epitope. Alternatively, the detectable group can beselected from a variety of other detectable groups or labels, such asradiolabels (e.g., ¹²⁵I, ³²P, ³⁵S) or a chemiluminescent or fluorescentgroup. Similarly, the detectable group can be a substrate, cofactor,inhibitor or affinity ligand.

2) Screening Test Agents Modulating Other Activities of KRS

Binding of a test agent to KRS provides an indication that the agent canbe a modulator of KRS. It also suggests that the agent may modulateactivity of laminin receptor to modulate cancer metastasis or tumor cellmigration. Thus, a test agent that binds to KRS can be further testedfor ability to modulate activity of laminin receptor

Alternatively, a test agent that binds to KRS can be further examined todetermine its activity on KRS. The existence, nature, and extent of suchactivity can be tested by an activity assay. Such an activity assay canconfirm that the test agent binding to KRS indeed has a modulatoryactivity on KRS. More often, such activity assays can be usedindependently to identify test agents that modulate activities of KRS(i.e., without first assaying their ability to bind to KRS). In general,such methods involve adding a test agent to a sample containing KRS inthe presence or absence of other molecules or reagents which arenecessary to test a biological activity of KRS and determining analteration in the biological activity of KRS. In addition to assays forscreening agents that modulate enzymatic or other biological activitiesof KRS, the activity assays also encompass in vitro screening and invivo screening for alterations in expression or cellular level of KRS.

Second Test Step: Screening Agents that Modulate Tumor Metastasis orTumor Cell Migration

Once a modulating agent has been identified to bind to KRS and/or tomodulate a biological activity (including cellular level) of KRS, it canbe further tested for ability to modulate tumor metastasis or tumor cellmigration. Modulation of tumor metastasis or tumor cell migration by themodulating agent is typically tested in the presence of KRS. When acell-based screening system is employed, KRS can be expressed from anexpression vector that has been introduced into a host cell.Alternatively, KRS can be supplied endogenously by the host cell in thescreening system.

The present invention also provides a method for screening an agentinhibiting an interaction between KRS and 67LR comprising:

(a) contacting a testing agent with KRS and laminin receptor (67LR) inthe presence of the testing agent; and

(b) testing whether the selected agent regulates an interaction betweenKRS and laminin receptor.

The said agent can be the things which stimulates or reinforceinteraction between KRS and laminin receptor (67LR), or in contrary, itcan be the things which inhibits or aggravates the interaction.

The (b) step can comprise detecting a relative change of the interactionlevel between KRS and 67LR polypeptide in the cell or the cell lysatethereof contacting the test agent compared to the interaction levelbetween KRS and 67LR in the cell or the cell lysate thereof withoutcontacting the test agent.

The method for identifying can be performed by any conventional methodknown in the art such as labeled in vitro protein-protein binding assays(in vitro full-down assays), EMSA (electrophoretic mobility shiftassays), immunoassays for protein binding, functional assays(phosphorylation assays, etc.), yeast two hybrid assay, assays ofnon-immune immunoprecipitations, Immunoprecipitation/The presentinventors stern blotting assays, immuno-co-localization assays.

For example, yeast two hybrid analyses may be carried out using yeastexpressing AIMP2 and p53, or parts or homologues of the proteins, fusedwith the DNA-binding domain of bacteria repressor LexA or yeast GAL4 andthe transactivation domain of yeast GAL4 protein, respectively (Kim, M.J. et al., Nat. Gent., 34:330-336, 2003). The interaction between AIMP2and p53 reconstructs a transactivator inducing the expression of areporter gene under the control by a promoter having a regulatorysequence binding to the DNA-binding domain of LexA or GAL4.

As described above, the reporter gene may be any gene known in the artencoding a detectable polypeptide. For example, chloramphenicolacetyltransferase (CAT), luciferase, β-galactosidase, β-glucosidase,alkaline phosphatase, green fluorescent protein (GFP), etc. may be used.If the interaction betweenAIMP2 and p53, or parts or homologues of theproteins is facilitated or enhance by a test agent, the expression ofthe reporter gene increases than under a normal condition. Conversely,if the interaction is inhibited or reduced by a test agent, the reportergene is not expressed or expressed less than under a normal condition.

Further, a reporter gene encoding a protein which enables growth ofyeast (i.e., if the reporter gene is not expressed, the growth of yeastis inhibited) may be selected. For example, auxotropic genes encodingenzymes involved in biosynthesis for obtaining amino acids ornitrogenous bases (e.g., yeast genes such as ADE3, HISS, etc. or similargenes from other species) may be used. If the expression of AIMP2 andp53, or parts or homologues of the proteins is inhibited or reduced by atest agent, the reporter gene is not expressed or less expressed.Accordingly, under such a condition, the growth of yeast is stopped orretarded. Such an effect on the expression of the reporter gene may beobserved with eyes or using devices (e.g., a microscope).

In addition, as results of analyzing overexpression of 67LR and KRSperformed to lung cancer or breast cancer patients, it is found that,relationship between overexpression of KRS and lung cancer or breastcancer is high in case of 67LR is over-expressed (table 1). Accordingly,the present invention provides a method for diagnosis of lung cancer orbreast cancer consisting of:

(a) analyzing overexpression of 67LR in a sample; and

(b) analyzing overexpression of LRS in the 67LR over-expressed sample.

Sampling for diagnosis and treatment for diagnosis and analysis ofoverexpression of 67LR and KRS may use molecular biological techniqueswhich are well known in the art and those are well described above.

Hereafter, the figures of the present invention will be described.

FIGS. 1 to 6 show specific interaction between human KRS and lamininreceptor. In FIG. 1, the interaction between full-length human KRS and37LRP/p40 was determined by yeast two hybrid assay. AIMP1 and AIMP2, thetwo components of the multi-ARS complex, were used as positive andnegative control, respectively. The positive interaction is indicated byblue colony formation on yeast medium containing X-gal. In FIG. 2, 37LRPwas synthesized by in vitro translation in the presence of [³⁵S]methionine and subjected to pull-down with GST-KRS or GST. 37LRPco-precipitated with GST-KRS was detected by autoradiography. In FIG. 3,The peptide regions of KRS and 37LRP involved in their interaction wasdetermined by yeast two hybrid assay as above. 37LRP contains 296 aminoacids in which the N-terminal cytoplasmic (amino acids 54-113) andC-terminal extracellular (amino acids 137-210) domains are divided bytransmembrane domain (amino acids 113-137). The N-terminal uniqueextension (about 70 amino acids) of human KRS (597 amino acids) isfollowed by OB-fold anticodon-binding (amino acids 70-214) and catalyticdomains (amino acids 220-574). In FIG. 4, A549 cells transfected withMyc-KRS were lyzed and subjected to immunoblot analysis with anti-Mycand anti-laminin receptor antibodies. Myc-KRS was immunoprecipitatedwith anti-Myc antibody and co-precipitated 67LR and 37LRP weredetermined by immunoblotting. For the specific blotting of 37LRP and67LR, the present inventors used polyclonal antibodies, H-141 and F-18(Santacruz), respectively (WCL: whole cell lysate). In FIG. 5, thelysates of Myc-KRS transfected A549 cells were subjected to Westernblotting with the indicated antibodies. The cells were separated tocytoplasmic and membrane fractions and immunoprecipitated with anti-Mycantibody and co-precipitation of 37LRP and 67LR was determined byWestern blotting. IgG was used as control. In FIG. 6, when treated with10 ug/ml of laminin for 1 hr, the present inventors confirmed that thebinding of 67LR and KRS was increased. To identify it,immunoprecipitation was performed with 67LR recognizing antibody (abcam,cat # ab2508), and the IgG level of the left is total IgG from a rabbitand was used as control. After SDS PAGE was performed, then was moved toPVDF membrane, and was performed immuno blot with KRS and 67LRrecognizing antibody respectively.

FIGS. 7 to 12 show that laminin-induced membrane translocation andphosphorylation of KRS. In FIG. 7, A549 cells were treated with laminin(10 ug/ml) and the level of 67LR, 37LRP and KRS was determined byWestern blotting at the indicated times. Hsp90 and Cadherin were used asmarkers for cytoplasm and membrane, respectively. In FIG. 8, A549 cellsuntreated or treated with laminin for 1 h were subjected toimmunofluorescence staining with anti-67LR (MLuC5, Santacruz, sc-59732)(red) and -KRS antibodies (green). In FIG. 9, A549 cells were treatedwith U73122 (U), staurosporin (ST) and LY294002 (LY) that inhibitPLC-gamma, PKC and PI3K, respectively, for 3 hr, then with laminin for 1hr and checked how these kinase inhibitors would affect the membrane andcytoplasmic level of 67LR and KRS. In FIG. 10, A549 cells weretransfected with Myc-KRS and incubated for 24 h. The cells were treatedwith the indicated chemicals and then with laminin as above. Myc-KRS wasimmunoprecipitated, and immunoblotted with anti-p-Thr, -Ser, and -Tyrantibodies. In FIG. 11, A549 cells were transfected with Myc-KRS andcultivated for 24 hr. The transfectants were pre-treated with LY294002for 3 hr and then treated with laminin for 1 h. Myc-KRS wasimmunoprecipitated and co-precipitation of 67LR was determined byWestern blotting. IgG was used as a control for immunoprecipitation. InFIG. 12, A549 cells were cultivated in the absence and presence oflaminin and LY294002 as indicated. EPRS (glutamyl-prolyl-tRNAsynthetase) was immunoprecipitated with its specific antibody (AbCam),and co-immunoprecipitation of KRS was determined by Western blotting(upper). The immune-depleted supernatant (ID) were subjected to Westernblottingting with anti-KRS and -EPRS antibodies.

FIGS. 13 to 17 show that KRS stabilizes membrane-bound 67LR. In FIG. 13,A549 cells were transfected with si-control or si-KRS and incubated inthe absence and presence of laminin. The cells were then separated tocytoplasm and membrane fractions, and the levels of 67LR and KRS in eachfraction were determined by Western blotting. Cadherin (cad) and hsp90were used as the markers for plasma membrane and cytoplasm,respectively. In FIG. 14, the membrane-bound 67LR level in A549 cellswas monitored by flow cytometry using anti-LR antibody (MLuC5). Thecells were transfected with empty vector or KRS plasmid and incubatedfor 24 h (upper). To see the effect of KRS suppression on 67LR level,the cells were transfected with si-KRS or si-control and incubated for48 h (lower). In FIG. 15, A549 cells transfected with EV or KRS wasselected with G418 for a week and cellular distribution of 67LR wasdetermined by immunofluorescence staining with anti-LR antibody (MLuC5).The membrane-located LR was highlighted with white arrows. In FIG. 16,A549 cells were treated with cycloheximide to inhibit de novo proteinsynthesis and the effect of KRS levels on 67LR level in membrane andcytoplasm was determined by Western blotting. In FIG. 17, the importanceof KRS for cellular stability of 67LR was determined by pulse-chaseexperiment. 293 cells were transfected with si-KRS or si-control andradioactive methionine was incorporated for 1 h. 67LR wasimmunoprecipitated with antibody specifically recognizing 67LR (F-18,Santacruz), separated by SDS-PAGE and autoradiographed. Suppression ofKRS with its specific siRNA was confirmed by Western blotting andtubulin is a loading control.

FIGS. 18 to 22 show that KRS enhances cell migration and cancermetastasis via 67LR. In FIG. 18, A549 cells were transfected with theindicated plasmids, incubated in the absence and presence of laminin andtheir effect on cell migration was determined by measuring the migratedcells in transwell chamber as described. The numbers of the cells passedthrough the membrane were counted and shown in each panel. Theexperiments were performed three times. In FIG. 19, the cells treated asabove were used to determine MMP-2 activity and level by zymography andWestern blotting, respectively. In FIG. 20, Breast carcinoma 4T-1 cellswere transfected with the indicated siRNA and subcutaneously injected tothe back of Balb/C mice. After 21 days, mouse lungs were isolated andtumor nodules over 1 mm in diameter were counted. In FIG. 21, twodifferent 4T-1 cells stably expressing exogenously introduced KRS (KRS-1and -2) were also inoculated as above and the tumor nodules were counted30 days after injection. 4T-1 cells with empty vector were used ascontrol. In FIG. 22, expression levels of KRS and 67LR in lung (upper)and breast (lower) cancer tissues were compared by immunohistochemicalstaining with their respective antibodies. 39 lung and 40 breast cancertissues were subjected to immunohistochemical staining with anti-KRS andanti-67LR antibodies and their expression levels were compared withthose in normal tissues (9 samples for each tissue). Shown here are therepresentative pairs of the same cancer patients demonstrating theoverexpression of KRS and 67LR. The result of statistical analysis forthe correlation between KRS and 67LR level is shown in table 1.

FIG. 23 shows membrane level of 67LR depends on KRS expression. 293cells transfected with the indicated plasmids were separated tocytoplasmic and membrane fractions, and the levels of 67LR, 37LRP andKRS in each fraction were determined by Western blotting with thecorresponding antibodies.

FIGS. 24 to 27 show effect of intracellular and extracellular KRS oncell migration, protein synthesis and cell cycle. In FIG. 24, Migrationof A549 cells incubated in the absence of laminin was determined wasdetermined by measuring the migrated cells in Transwell chamber. In FIG.25, to see the chemotactic activity of KRS, the serum-free mediumcontaining the indicated KRS concentration was placed in the lowerchamber and A549 cells were incubated in the upper chamber in Transwellchamber. After 6 hr of incubation, the migrant were counted. In FIG. 26,KRS level in A549 cells was down- and up-regulated by introduction ofsiRNA and exogenous KRS (lower panels of FIGS. 26 and 27). Thetransfected cells were incubated for 48 and 24 hr, respectively andstarved in methionine-free medium for 1 hr and radioactively labeledmethionine was incorporated for 2 hr. After washing, the cells wereincubated for 4 h and lyzed in 0.5% triton X-100 lysis solution, andincorporated radioactivity was determined by liquid scintillationcounting. In FIG. 27, A549 cells transfected as indicated were fixed andstained with propidium iodide, and their cell cycle was determined byflow cytometry.

FIGS. 28 to 30 show the effect of KRS suppression on cancer metastasis.In FIG. 28, the effect of si-KRS and -DRS on the expression of theirtarget proteins was determined by Western blotting. Tubulin was used asloading control. In FIG. 29, the siRNA transfected cells (1×10⁶) wereinjected as described in methods and the effect of KRS and DRSsuppression on primary tumor growth was determined by measuring tumorweight and volumes 21 days after inoculation. Each group contained 5mice. In FIG. 30, the lungs isolated above were fixed in 10% formalin.The number and size of metastatic tumor nodules were shown.

FIGS. 31 to 33 show the effect of KRS overexpression on cancermetastasis. In FIG. 31, overexpression of KRS-1 and -2 cell lines wasdetermined by Western blotting. In FIG. 32, the effect of KRSoverexpression on primary tumor growth was also compared as above. InFIG. 33, the effect of KRS overexpression on cancer metastasis wasdetermined 30 days after inoculation. Each group contained 4 mice.

DESCRIPTION OF DRAWINGS

FIG. 1 is the result confirming the interaction between human KRS and37LRP/p40 using yeast two hybrid assay.

FIG. 2 is the result confirming the interaction between human KRS and37LRP using pull-down assay.

FIG. 3 is the result confirming the region of interaction between humanKRS and 37LRP.

FIG. 4 is the result of immunoblot analysis to confirm the binding ofKRS to 67LR and 37LRP in A549 cells transfected with Myc-KRS usinganti-Myc and anti-laminin receptor antibodies.

FIG. 5 is the result of Western blotting analysis to confirm the bindingof KRS to 67LR and 37LRP in the lysates of Myc-KRS transfected A549cells.

FIG. 6 is the result of immunoprecipitation to confirm the binding of67LR and KRS depending on the treatment with laminin.

FIG. 7 is the result of Western blotting to confirm the level of 67LR,37LRP and KRS depending on the treatment with laminin.

FIG. 8 is the result of immunofluorescence staining to examine anexpression level of 67LR and KRS depending on the treatment with lamininin A549 cells.

FIG. 9 is the result confirming the effect of kinase inhibitors on themembrane and cytoplasmic expression level of 67LR and KRS.

FIG. 10 is the result of immunoblot to measure the phosphorylation levelin KRS expressing-A549 cell when laminin and kinase inhibitor weretreated using anti-p-Thr, -Ser, and -Tyr antibodies aboutphosphorylation.

FIG. 11 is the result of Western blotting to determine the binding ofphosphorylated KRS to 67LR in the KRS expressing A549 cells.

FIG. 12 is the result of Western blotting to confirm the effect oflaminin on binding of KRS and EPRS.

FIG. 13 is the result of Western blotting to confirm 67LR and KRS levelsin A549 cell transfected with si-control or si-KRS.

FIG. 14 is the result of flow cytometry to confirm the membrane-bound67LR level in A549 cells.

FIG. 15 is the result of immunofluorescence staining to confirm cellulardistribution of 67LR in A549 cell with EV (empty vector) or KRS.

FIG. 16 is the result of Western blotting to confirm the effect of KRSlevels on 67LR level in membrane and cytoplasm in A549 cells inhibitedde novo protein synthesis.

FIG. 17 is the result of pulse-chase experiment confirming theimportance of KRS for cellular stability of 67LR.

FIG. 18 is the result confirming the effect on cell migration when theexpressions of KRS and/or 67LR are inhibited.

FIG. 19 is the result of zymography and Western blotting to determineMMP-2 activity and level when the expressions of KRS and/or 67LR areinhibited.

FIG. 20 shows the number of tumor nodules when the expressions of KRSare inhibited in mouse transplanted with 4T-1 cells.

FIG. 21 shows the number tumor nodules when the expressions of KRS areenhanced in mouse transplanted with 4T-1 cells.

FIG. 22 is the result of immunohistochemical staining to confirm theexpression levels of KRS and 67LR in lung and breast cancer tissues.

FIG. 23 is the result of Western blotting to confirm membrane level of67LR depends on KRS expression.

FIG. 24 is the result of measuring migration of A549 cells in theabsence of laminin.

FIG. 25 is the result of measuring the chemotactic activity of KRS incell migration.

FIG. 26 is the result of confirming KRS level and total proteinsynthesis in A549 cells by introduction of siRNA and exogenous KRS.

FIG. 27 is the result of confirming KRS level and cell cycle in A549cells by introduction of siRNA and exogenous KRS.

FIG. 28 is the result of Western blotting to confirm the effect ofsi-KRS and si-DRS on the expression of their target proteins.

FIG. 29 is the result of confirming the effect of KRS and DRSsuppression on primary tumor growth in tumor cell transplantation.

FIG. 30 is the result of confirming the number and size of metastatictumor nodule in tumor cell transplantation.

FIG. 31 is the result of Western blotting to confirm overexpression ofKRS in KRS-1 and -2 cell lines.

FIG. 32 is the result of confirming the effect of KRS overexpression onprimary tumor growth in tumor cell transplantation.

FIG. 33 is the result of confirming the number and size of metastatictumor nodule in tumor cell transplantation.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail byexamples. It is to be understood, however, that these examples are forillustrative purpose only and are not constructed to limit the scope ofthe present invention.

Experimental Method 1. Cell Culture and Materials

A549 and HEK293 were purchased from ATCC. Mouse mammary carcinoma 4T-1cell line was kindly provided by Dr. Seong Jin Kim (Gachun MedicalSchool). RPMI (for A549 and 4T-1 cells) and Dulbecco's Modified EagleMedium (for the other cell lines), containing 10% fetal bovine serum and1% antibiotics were used for cell cultivation. pcDNA3.1 encoding 37LRPwas a kind gift from Dr. Hirofumi Tachibana (Kyushu University).Myc-tagged human KRS and DRS werecloned at the EcoRI/XhoI site ofpcDNA3. Murine KRS cDNA was obtained by RT-PCR and cloned atHindIII/XhoI site of pcDNA3.1. siRNAs targeting murine and human KRS andDRS were purchased from Invitrogen. Sequences for siRNAs would beprovided upon request. Gene porter (GTS) and Lipofectamine 2000(Invitrogen) were used as transfection reagent. LY294002, U73122 andstaurosporin were purchased from Calbiochem, and cycloheximide andlaminin (Engelbreth-Holm-Swarm murine sarcoma) from Sigma.

2. Immunoprecipitation and the Present Inventorsstern Blot.

The cells were lysed with 20 mM Tris-HCl (pH 7.4) buffer containing 150mM NaCl, 0.5% TritonX-100, 0.1% SDS, and protease inhibitor. The proteinextracts were incubated with normal IgG and protein G agarose for 2 hrand then centrifuged to remove nonspecific IgG binding proteins. Thepresent inventors then mixed the supernatants with purified 67LRantibody (F-18, Santacruz), incubated for 2 hr at 4° C. with agitation,and added protein A agarose. After washing three times with the ice-coldlysis buffer, the precipitates were dissolved in the SDS sample bufferand separated by SDS-PAGE. To determine the binding of KRS and LR indifferent cell fractions, the present inventors transfectedpcDNA3.1-Myc-KRS and separated the plasma membrane and cytoplasmicfractions using the proteoextract kit (Calbiochem) following themanufacturer's instruction, and co-immunoprecipitation was performed asabove. To analyze protein levels, the proteins extracted from the cellswere separated by 10% SDS-PAGE. Anti-LR antibody (Abcam, ab2508) wasused for simultaneous immunoblotting of 37LRP and 67LR unless specified.Antibodies for hsp90 and Pan-cadherin were purchased from Santacruz.

3. Flow Cytometry

To address cell cycle, the cultivated cells were transfected or treatedwith the indicated vector or chemicals, fixed with 70% ethanol for 1 hrat 4° C. and washed with ice-cold PBS two times. The cells were thenstained with propidium iodide (50 ug/ml), sodium citrate 0.1%, NP40 0.3%and RNaseA (50 ug/ml) for 40 min and subjected to flow cytometry (FACSCalibur, Beckton-Dickinson). For each sample, 20,000 cells were analyzedusing Cell Quest Pro software. For analysis of amount of 67 kD LR oncell surface, 1×10⁶ cells were incubated IgG or anti-LR antibody (MLuC51 ug) recognizing extracellular domain of 67LR and then with FITCsecondary antibody. After washing with PBS, the samples were scanned byFACS.

4. Immunofluorescent and Immunohistochemical Staining

A549 cells on a 9 mm cover slip were fixed with 70% methyl alcohol andwashed briefly with cold phosphate buffer saline (PBS). After incubationwith blocking buffer containing 1% CAS, 3% BSA and 0.5% tritonX-100 for30 min, the cells were incubated with antibody against KRS (Abcam), andMLuC-5 (Santacruz) for 1 hr. Alexa488 and 568 (Invitrogen) were thenadded for 30 min at room temperature. After washing with cold PBS for 30min, specimens were observed by laser-scanning microscopy. The tissuearray slides for breast and lung cancer were purchased fromSuper-Biochip (Korea) and subjected to immunohistochemical staining todetermine the expression level of 67LR and KRS with their respectiveantibodies as described (Park, S. G. et al. Human lysyl-tRNA synthetaseis secreted to trigger pro-inflammatory response, Proc. Natl. Acad. Sci.USA 102, 6356-6361 (2005)). Statistical analyses were performed usingthe Pearson χ² test and Student t test to evaluate the correlationbetween 67LR and KRS expression. P values <0.05 were consideredsignificant. All statistical analyses were performed using SPSS v11.5software (SPSS, Chicago, Ill.).

5. Pulse-Chase Experiment

293 cells were transfected with si-KRS or si-control (Invitrogen) usinglipofectamine 2000. The cells werethen incubated with methionine-freemedium for 1 hr, and [³⁵S] methionine (50 □Ci/ml) was added andincubated for 1 h. After washing off the radioactive methionine withfresh medium, 67LR was immunoprecipitated with its specific antibody(Santacruz), separated by 12% SDS-PAGE and subjected to autoradiagraphyusing BAS (FLA-3000, FujiFilm). The amount of 67LR was quantified byMulti-gauge program (V3.0, FujiFilm).

6. Yeast Two Hybrid Analysis

cDNAs encoding different fragments of human KRS were obtained by PCRwith the corresponding primers. The PCR product for KRS was digestedwith EcoRI and XhoI, and ligated the corresponding sites of pEG202 (forthe construction of LexA-fusion proteins) and pJG4-5 (for theconstruction of B42-fusion proteins). The cDNAs encoding 37LRP fragmentswere kindly provided from Dr. Barbara J. Ballermann (University ofAlberta), and they were subcloned at EcoRI and XhoI sites of pJG4-5. Theinteractions between the two fusion protein series were analyzed by theformation of blue colonies on the X-gal-containing yeast medium.

7. In Vitro Binding Assay.

The present inventors expressed GST-KRS or GST in Escherichia coliRosetta (DE3) strain, mixed the protein extracts withglutathione-Sepharose in the PBS buffer containing 1% Triton X-100 and0.5% N-laurylsarcosine at 4° C. for 2 h. The present inventorssynthesized human 37LRP by in vitro translation in the presence of [³⁵S]methionine using pcDNA3-37LRP as the template using TNT Quick coupledTranscription/Translation system (Promega). The synthesized 37LRP wasadded to the GST protein mixtures above, incubated at 4° C. for 4 hrwith rotation in the PBS buffer containing 1% Triton X-100, 0.5%N-laurylsarcosine, 1 mM DTT, 2 mM EDTA and 300 □M phenylmethylsulfonylfluoride, and washed six times with the same buffer containing 0.5%Triton X-100. The present inventors then eluted the proteins bound toSepharose beads with the SDS sample buffer, separated by SDS-PAGE andautoradiographed.

8. Cell Migration Assay

Cell migration was determined by using 24-Transwell chambers withpolycarbonate membranes (8.0 um pore size, Costar) as previouslydescribed (Park, S. G. et al. Human lysyl-tRNA synthetase is secreted totrigger pro-inflammatory response, Proc. Natl. Acad. Sci. USA 102,6356-6361 (2005)). A549 cells were suspended in serum-free RPMI andadded to the upper chamber at 1×10⁵ cells per well. Each of the purifiedhuman KRS at the indicated concentrations, laminin (10 μg/ml) or gelatin(10 μg/ml) was placed in the lower well, and the cells were allowed tomigrate for 6 hr at 37° C. in CO₂ incubator. The cells were fixed with70% methyl alcohol in PBS for 30 min and washed with PBS three times.The cells were stained with hematoxylin (Sigma) for 10 min and washedwith distilled water. The non-migrant cells were removed from the upperface of the membrane with a cotton swab. The membranes were excised fromthe chamber and mounted with Gel Mount (Biomeda, Foster City, Calif.).The migrant cells (those attached to the lower face of the membrane)were counted at four randomly selected scopes in high power fields(×20).

9. Zymography

A549 cells transfected with the plasmids encoding the indicated siRNAsand recombinant KRS (or DRS) were incubated for 48 and 24 hr,respectively, and were seeded (1×10⁵ cells/well) in RPMI containing 10%FBS. After starving the cells in serum-free RPMI for 2 hr, laminin wasadded and incubated for 24 hr at 10 μg/ml. 20 □l of the culture mediumwas mixed with 5×FOD buffer (0.125M Tris-HCl, pH 6.8, containing 4% SDS,20% glycerol and 0.01% bromophenol blue) and subjected to 10% SDS-PAGEcontaining 1 mg/ml of gelatin. The gel was washed with 2.5% Triton X-100twice for each 20 min, then with distilled water twice for each 20 minand incubated with the reaction buffer (50 mM Tris-HCl, pH 7.5,containing 10 mM CaCl2, 150 mM NaCl, 1 □M ZnCl₂, 1% Triton X-100, 0.002%sodium azide) for 24 h at 37° C. The gel was washed with distilled waterand stained with Coomassie blue R250 and destained with 35% methanol.

10. Cancer Metastasis Experiment In Vivo

Mouse mammary carcinoma 4T-1 cells were transfected with si-KRS-DRS orsi-control and incubated for 24 hr. The cells (1×10⁶) weresubcutaneously inoculated into the back of 6-week old female Balb/cmice. The effect of siRNAs to their target expression was tested in theremaining cells 48 hr after transfection and also in the primary tumorsfrom 3 to 10 days at 2 days intervals after inoculation by Westernblotting with their corresponding antibodies. The growth of tumor wasmonitored by measuring tumor size three times weekly. The whole bodyweights were also measured at the same time. The mice were sacrificed 21days after inoculation and the primary tumors and lungs were excisedfrom the animals. The lungs were fixed in 10% formalin for twenty fourhours. The number and size of metastatic tumor nodules on lungs werecounted, and tumor nodules of larger than 1 mm in diameter were recordedseparately. The primary tumors were also weighed. To examine the effectof KRS overexpression on cancer metastasis, murine KRS vector or emptyvector were transfected into 4T-1 cells and stable transfectants wereselected by the incubation in the presence of G418 for 3 weeks. Thepresent inventors then picked up several single colonies and comparedKRS expression level by Western blotting. Two different colonies (KRS-1and -2) expressing KRS at higher level than the control cells werechosen and used for inoculation. All the procedures were performed asabove except that the mice were sacrificed 30 days after inoculation.

Experimental Result and Discussion

The specific interaction between full-length KRS and 37LRP was confirmedby yeast two hybrid assay. LexA-KRS generated blue colonies when pairedwith B42-37LRP as well as AIMP2, the known partner of KRS (Kim, J. Y. etal. p38 is essential for the assembly and stability of macromoleculartRNA synthetase complex: Implications for its physiologicalsignificance, Proc. Natl. Acad. Sci. USA 99, 7912-7916 (2002)), but notwith AIMP1 (FIG. 1). For in vitro binding assay, [³⁵S]methionine-labelled 37LRP was mixed with either GST-KRS or GST,precipitated with glutathione-Sepharose and subjected toautoradiography. 37LRP was co-precipitated with GST-KRS, but not withGST (FIG. 2). Deletion mapping by yeast two hybrid assay determined thatthe N-terminal extension of human KRS and the C-terminal extracellulardomain of LR are involved in their association (FIG. 3).

Since cytoplasmic 37LRP is converted to membrane-embedded 67LR, thepresent inventors checked whether KRS would bind differently between37LPR and 67LR. Myc-KRS was introduced into lung carcinoma A549 cellsand immunoprecipited with anti-Myc antibody. The present inventors sternblotting of the whole cell lysate demonstrated that 67LR exists at lowerlevel than 37LRP (FIG. 4 right). Nonetheless, Myc-KRS predominantlybound to 67LR than 37LRP (FIG. 4 left). The present inventors thenseparated A549 cells into cytoplasmic and plasma membrane fractions anddetermined the interaction of Myc-KRS with 67LR and 37LRP. 37LRP and67LR were mainly detected at cytoplasm and plasma membrane, respectively(FIG. 5 right), while KRS existed at both fractions although a majorportion was observed at cytoplasm. When both fractions were subjected toimmunoprecipitation with anti-Myc antibody, the membrane-bound 67LR wasmainly co-precipitated with KRS although low amount of 37LRP incytoplasm was also precipitated (FIG. 5 left), indicating preferentialinteraction between membrane-resident 67LR and KRS.

The present inventors then investigated whether cellular distribution ofKRS is changed by laminin treatment in A549 cells by cell fractionationand immunofluorescence staining. After laminin treatment, membrane levelof KRS and 67LR was gradually increased with little changes in thecytoplasmic KRS and 37LRP level or their expression (FIG. 7 and data notshown) Immunofluorescence staining also demonstrated the shift of 67LRand KRS toward membrane side by laminin treatment (FIG. 8, red andgreen, respectively). The present inventors then investigated whethermembrane translocation of KRS involves post-translational modification.A few different kinases such as phosphoinositide 3-OH kinase(PI3K)(Shaw, L. M., Rabinovitz, I., Wang, H. H., Toker, A. & Mericurio.A. M. Activation of phosphoinositide 3-OH kinase by the alpha6beta4integrin promotes carcinoma invasion. Cell 91, 949-960 (1997)), proteinkinase C (PKC)(Li, Y. Q. et al. Protein kinase C mediates the signal forinterferon-gamma mRNA expression in cytotoxic T cells after theiradhesion to laminin Immunology 93, 455-461 (1998)) and phospholipaseC-gamma (PLC-gamma)(Vossmeyer, D., Hofmann, W., Loster, K., Reutter, W.& Danker, K. Phospholipase C-gamma binds alphalbetal integrin andmodulates alpha1beta1 integrin-specific adhesion. J. Biol. Chem. 277,4636-4643 (2002); Kanner, S. B., Grosmaire, L. S., Ledbetter, J. A. &Damle, N. K. Beta 2-integrin LFA-1 signaling through phospholipaseC-gamma 1 activation. Proc. Natl. Acad. Sci. USA 90, 7099-7103 (1993))are known to be activated by laminin. To see whether any of thesekinases are involved in laminin-dependent membrane translocation of KRS,the present inventors blocked each of these kinases with their specificinhibitors, and checked how these treatments would affect the membranetranslocation of KRS. Laminin-dependent increase of KRS and 67LR in themembrane fraction was blocked in the presence of LY294002, the PI3Kinhibitor whereas the cells treated with U73122 or staurosporin stillshowed laminin-dependent induction of 67LRS as the control cells (FIG. 9upper and data not shown). None of these kinases affected thecytoplasmic level of KRS (FIG. 9 lower). These results imply that PI3Kshould be involved in laminin-induced phosphorylation of KRS. In fact,phosphorylated KRS at threonine and serine, but not at tyrosine wasincreased by laminin treatment, but blocked in the presence of LY294002while staurosporin did not give any effect (FIG. 10). The presentinventors then checked whether the laminin-induced phosphorylation ofKRS would be necessary for its interaction with 67LR. The treatment ofLY294002 suppressed the laminin-induced association of KRS with 67LR(FIG. 11). Since cytoplasmic KRS is anchored to the multi-ARS complex,the present inventors also checked whether laminin-dependentphosphorylation of KRS would affect its association with the multi-ARScomplex by co-immunoprecipitation of KRS with glutamyl-prolyl-tRNAsynthetase (EPRS), another enzyme component of the complex. In theabsence of LY compound, laminin treatment decreased the association ofKRS with EPRS with the simultaneous increase of KRS in immuno-depletedsoluble fraction (FIG. 12 left lanes in upper and lower panels). Incontrast, the KRS binding to EPRS was not affected by laminin treatmentwhen the cells were pre-treated with LY294002 compound (FIG. 12 rightlanes in upper and lower panels), suggesting that the phosphorylation ofKRS is necessary for the laminin-dependent dissociation of KRS from thecomplex.

The present inventors then checked whether KRS would affect the membranelevel of 67LR in A549 cells. The 67LR level was increased by laminin butthe laminin effect was abolished when KRS was suppressed with itsspecific siRNA (FIG. 13 left), indicating the importance of KRS inlaminin-dependent enhancement of 67LR. The present inventors alsomonitored membrane-bound 67LR by flow cytometry. The membrane 67LR levelincreased and decreased when the cells were transfected with KRS andsi-KRS, respectively (FIG. 14). Cellular distribution of lamininreceptor was compared between A549 cells transfected with EV or KRS byimmunoflurescence staining Laminin receptor was more densely stained inplasma membrane regions in KRS-overexpressing cells compared to that inthe control cells (FIG. 15). The positive correlation between KRS and67LR level was further confirmed by measuring 67LR in membrane andcytoplasm according to the variation of KRS level (FIG. 23).

The present inventors then investigated how KRS enhances membrane level67LR. KRS can stimulate the 67LR synthesis through transcription orconversion from 37LRP. However, transfection of KRS did not increase LRtranscription (data not shown), excluding its potential role in theregulation of LR transcription. Besides, since KRS showed poor bindingto 37LRP in cytoplasm (FIGS. 4 and 5), it is unlikely that it stimulatesthe conversion process of 37LRP to 67LR. The present inventors alsochecked whether KRS would mediate fatty acylation of 37LRP since thismodification is known to be prerequisite for the conversion of 37LRP to67LR (Landowski, T. H., Dratz, E., A. & Starkey, J. R. Studies of thestructure of the metastasis-associated 67 kDa laminin binding protein:fatty acid acylation and evidence supporting dimerization of the 32 kDagene product to form the mature protein. Biochemistry 34, 11276-11287(1995); Buto, S. et al. Formation of the 67-kDa laminin receptor byacylation of the precursor. J. Cell. Biochem. 69, 244-251 (1998)). Inour assay, KRS did not affect the fatty acylation of 37LRP either (datanot shown). Since KRS can extend cellular stability of themembrane-bound 67LR, the present inventors checked whether KRS wouldinterfere with endocytosis of membrane-bound 67LR. To see thispossibility, the present inventors arrested de novo protein synthesiswith cycloheximide and examined whether KRS would affect the 67LR levelin membrane and cytoplasm. When KRS expression was suppressed with itsspecific siRNA, the membrane level of 67LR was decreased with concurrentincrease of 67LR in the cytoplasmic fraction (FIG. 16 left). Conversely,overexpression of KRS increased the membrane level of 67LR as above(FIG. 16 right). Based on these results, KRS appears to extend themembrane residency of 67LR by blocking its re-entry to cytoplasm. Thepresent inventors further investigated the effect of KRS on turnover of67LR by pulse-chase experiment. Nascent protein synthesis was labeledwith radioactive methionine and then blocked with cycloheximide. Then,disappearance of 67LR was monitored by autoradiography at time interval.67LR was rapidly decreased when KRS was suppressed with its siRNAwhereas its level was well sustained in si-control cells during thistime frame (FIG. 17). Thus, KRS seems to extend half life of 67LRthrough its association with 67LR in plasma membrane, thereby inhibitingendocytosis of 67LR although degradation process of 67LR needs furtherinvestigation.

The present inventors then investigated whether KRS expression levelwould affect laminin-dependent A549 cell migration using Transwellmembrane assay. Migration of the control cells was enhanced about 6 foldin average by laminin treatment (FIG. 24 and FIG. 18). However, thelaminin-dependent cell migration was reduced when KRS was suppressedwith its specific siRNA (FIG. 18, si-control and si-KRS). Conversely,KRS overexpression further augmented cell migration induced by laminintreatment (FIG. 18, EV and KRS). However, the KRS effect on cellmigration was diminished when laminin receptor was suppressed with itssiRNA (FIG. 18 si-LR, bottom panel). Since KRS is also secreted in somecancer cells as cytokine (Park, S. G. et al. Human lysyl-tRNA synthetaseis secreted to trigger pro-inflammatory response, Proc. Natl. Acad. Sci.USA 102, 6356-6361 (2005)), the present inventors checked whetherextracellular KRS would affect cell migration. When A549 cells weretreated with purified KRS at different concentration, cell migration waslittle affected (FIG. 25), excluding the extracellular effect of KRS inthis assay. Besides, cellular protein synthesis and cell cycle were notinfluenced by suppression or overexpression of KRS during the period ofexperiments (FIGS. 26 and 27), indicating that KRS-dependent cellmigration did not result from its effect on these processes either.Since laminin treatment results in the activation of MMP-2 (matrixmetllo-proteinase-2)(Givant-Horwitz, V., Davidson, B. & Reich, R.Laminin-induced signaling in tumor cells; the role of the M(r) 67,000laminin receptor. Cancer Res. 64, 3572-3579 (2004)), the presentinventors checked the effect of KRS on the laminin-dependent activationof MMP-2 using in vitro zymography assay. MMP-2 activity was enhanced bylaminin, which was blocked in the presence of si-KRS (FIG. 19 left), butfurther enhanced by overexpression of KRS (FIG. 19 right). Theexpression level of MMP-2 was not affected by KRS (FIG. 19 bottom).

Since KRS can induce cell migration via 67LR that is implicated incancer metastasis, the present inventors examined whether cancermetastasis would be also affected by the expression level of KRS using4T-1 mouse mammary carcinoma cells that are highly metastatic to lung.The present inventors suppressed either KRS or DRS (aspartyl-tRNAsynthetase), another component of multi-ARS complex, with their specificsiRNAs and compared how down-regulation of KRS and DRS would affectcancer metastasis. After confirming the suppression effect of si-KRS and-DRS by Western blotting (FIG. 28), each of these cells and the cellswith si-control was subcutaneously injected into the back skin of Balb/cmice. All of the three injected cells developed tumors of similar thepresent inventor sight and volume (FIG. 29), suggesting that KRS leveldid not affect the growth of primary tumors. Lungs were isolated 21 daysafter inoculation and the numbers of the metastatic tumor nodules(larger than 1 mm in diameter) were compared between the three groups.The number of the metastatic nodules was significantly decreased by thesuppression of KRS compared to those obtained from the control andDRS-suppressed cells (FIG. 20 and FIG. 30). Conversely, the presentinventors examined whether overexpression of KRS would enhance cancermetastasis using the same method as above. The present inventors firstestablished 4T-1 cell lines stably overexpressing KRS by transfection ofthe KRS-encoding plasmid and G418 screening. KRS overexpression in theestablished cell lines were confirmed by Westhern blotting, and thepresent inventors selected the two different cells (KRS-1 and KRS-2)expressing KRS at higher amount than those transfected with empty vector(FIG. 31). These cells also generated primary tumors of similar weightand size (FIG. 32). When the present inventors examined the lungs in 30days after inoculation of the cells, both of the KRS-overexpressingcells generated more nodules compared to the control cells (FIG. 21 andFIG. 33). All of these results suggest that KRS can induce cancermetastasis in vivo.

Since cancer-specific overexpression of laminin receptor has beenfrequently observed (Fontanini, G. et al. 67-Kilodalton laminin receptorexpression correlates with worse prognostic indicators in non-small celllung carcinomas. Clin. Cancer Res. 3, 227-231 (1997), Viacava, P. et al.The spectrum of 67-kD laminin receptor expression in breast carcinomaprogression. J. Pathol. 182, 36-44 (1997), the present inventorsanalyzed whether overexpression of 67LR is also associated with that ofKRS by immunohistochemical staining of 67LR and KRS in lung and breastcancers as the examples. Among the 39 examined lung cancer tissues, 67LRoverexpression was observed in 21 cases (54%), in which KRS level wasalso increased in 19 cases (about 90%) (Table 1 and FIG. 22 upper).Likewise, the 21 cases out of the 40 examined breast cancer patientsshowed 67LR overexpression. In these cases, all 21 cases also showedincreased level of KRS (Table 1 and FIG. 22 lower). In both cases, thetight linkage between the expressions of the two proteins is shownalthough it is to be determined whether their co-expression in cancer isactually implicated in metastasis.

TABLE 1 67LR 67LR lung cancer Normal Overexpression Total KRS Normal 102 12 KRS Overexpression 8 19 27 Total 18 21 39 * fisher's exact test p =0.001 67LR 67LR Breast cancer Normal Overexpression Total KRS Normal 5 05 KRS Overexpression 14 21 35 Total 19 11 40 * fisher's exact test p =0.018At this time, it may be referred as followed regarding the table 1. Thetable 1 is the correlation between 67LR and KRS expression in cancertissues. To test whether expression level of 67LR is associated withthat of KRS, tissue microarrays of lung and breast cancer patients weresubjected to immunohistochemical staining with their respectiveantibodies, and the relative expression levels of the two proteins weredetermined. MLuC5 antibody was used for immunodetection of 67LR.Expression level was determined by staining intensity of the specimenand classified into 4 groups (score 0, 1, 2, and 3). In the finalevaluation, the samples were divided into normal (with a score 0 or 1)and overexpression group (with a score 2 or 3). Statistical analyseswere performed using the Pearson χ² test and Student t test to evaluatethe correlation between 67LR and KRS expression. P values <0.05 wereconsidered significant. All statistical analyses were performed usingSPSS v11.5 software (SPSS, Chicago, Ill.).

Many translational factors including ribosomal components arepleiotropic (Wool, I. G. Extraribosomal functions of ribosomal proteinsTrends Biochem. Sci. 21, 164-165 (1996)) and associated with varioustumorigenesis (Lee, S. W., Kang, Y. S. & Kim, S Multi-functionalproteins in tumorigenesis: Aminoacyl-tRNA synthetases and translationalcomponents. Curr. Proteomics 3, 233-247 (2006)). Here the presentinventors demonstrated that two translational factors, KRS andp40/37LRP, work together for cell migration and cancer metastasis invivo (FIGS. 18 and 22). At this moment, the present inventors do notknow whether the potential association of these two proteins is theevolutionary coincidence or has another physiological reason in proteinsynthesis that needs to be understood in the future. Among thecomponents of the multi-ARS complex, KRS is the most stable protein andrequired for the stability of other components (Han, J. M. et al.Hierarchical Network between the components of the multi-tRNA synthetasecomplex: Implications for complex formation. J. Biol. Chem. 281,38663-38667 (2006)), implying its potential to stabilize the associatedproteins. Here the present inventors showed that KRS also extendscellular stability of 67LR (FIG. 17).

The association of KRS with 67LR may have different functionalimplications. Under physiological condition, a portion of cytoplasmicKRS is phosphorylated and mobilized to the plasma membrane by variousgrowth-stimulatory or survival signals to bind 67LR that mediateslaminin signal. In cancer cells, membrane level of KRS could beabnormally enhanced either due to its overexpression or its constitutivemembrane translocation resulting from the hyperactivated upstreamkinases such as PI3K. Perhaps, these excess KRS could be driven to theplasma membrane that is either recruited to 67LR or secreted. Inaddition, it is worth noting that the deregulated activation of PI3K isoften associated with tumor growth and metastasis (Wymann, M. P. &Marone, R. Phosphoinositide 3-kinase in disease: timing, location, andscaffolding. Curr. Opin. Cell Biol. 17, 141-149 (2005)), and lamininpromotes cancer invasion via PI3K (Baba, Y. et al. Laminin-332 promotesthe invasion of oesophageal squamous cell carcinoma via PI3K activation.Br. J. Cancer 98, 974-980 (2008)). The constitutive activation of PI3Kmay lead to the phosphorylation of KRS that would be mobilized to themembrane. Either or both of these conditions could contribute to theincrease of 67LR in the plasma membrane, thereby amplifying the lamininsignaling for cancer metastasis. Much investigation is being made tocontrol metastatic spread of cancer. In this regard, KRS activity incancer metastasis via 67LR may provide a previously unexplored windowfor cancer diagnosis and therapy.

As can be seen from the foregoing, the present inventors disclosed thatthe inventive KRS interacts with 67LR through translocation of KRS intoplasma membrane, and so enhances tumor (or cancer) cell migration,thereby having an effect on cancer metastasis. In addition, we alsodisclosed that KRS overexpression or inhibition of KRS expression canmodulate tumor (or cancer) cell metastasis through in vivo experimentsusing mice. Accordingly, cancer metastasis and cancer cell migration maybe controlled using the inventive KRS, further the cellular metabolismrelated to laminin receptor (67LR) of plasma membrane may be controlled.The relationship between KRS and laminin receptor disclosed in thepresent invention may be very useful for treatment, prevention and/ordiagnosis of various disease related to thereof.

1. A method for screening an agent inhibiting an interaction between KRSand 67LR comprising: (a) contacting a testing agent with KRS and lamininreceptor (67LR) in the presence of the testing agent; and (b) testingwhether the selected agent regulates an interaction between KRS andlaminin receptor.